Ionization method and apparatus using electrospray, and analyzing method and apparatus

ABSTRACT

It is arranged so that living tissue that has not been pretreated can be adopted as a sample of interest. A sample is introduced into at least a tip portion of a hollow insulated sample holder  11  having a small hole  11   a  in the tip portion. A slender metal wire  12 , which has been inserted into the sample holder  11  from the rear, is projected outwardly from the sample holder  11  through the hole  11   a  while being brought into contact with the sample. A high voltage is applied to the slender metal wire  12  at least in a portion of a time period in which the tip of the slender metal wire  12  is being projected outwardly from the hole  11   a , thereby ionizing, by electrospray, the sample adhering to the tip of the slender metal wire  12 . The ions are introduced into an analyzing apparatus and are analyzed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an ionization method and apparatususing electrospray, and to an analyzing method and apparatus.

2. Description of the Related Art

A typical method of imaging mass spectrometry for dealing withbiological samples and industrial products and the like is massspectrometry using matrix-assisted laser desorption ionization (MALDI).With this method, a pretreatment, namely the preparation of a MALDIsample, is required.

The molecular analysis of live single cells has been gaining momentum inrecent years, and nano-electrospray (ESI) mass spectrometry has beenproposed as an effective analytical method for this purpose.

See Mizuno, Tsuyama, Harada and Masujima “Live single-cell video-massspectrometry for cellular and subcellular molecular detection and cellclassification”, J. Mass Spectrom. 2008; 43: 1692-1700.

According to this method, a cell of interest or cellular solutionthereof is drawn into the end of a glass ESI tip (a capillary), the endhaving an opening diameter (inner diameter) on the micron order, anionization solvent (for example, acetonitrile containing 0.5% formicacid for the positive-ion mode, or 0.5% ammonia water for thenegative-ion mode) is applied to the interior of the ESI tip to achievedilution by 10⁶ times or more, then the diluted cellular sample isionized by electrospray and the result introduced to an analyzingapparatus. With this method, the solvent also undergoes massspectrometry as a matter of course, and an ion signal ascribable tomolecules resulting from the solvent shows up in the mass spectrometryspectra.

SUMMARY OF THE INVENTION

The present invention provides an ionization method and apparatus thatenable living cells or the like, which have not been pretreated, to beadopted as a sample of interest (inclusive of a single cell, the bodyfluid of a living animal, etc.).

The present invention further provides an ionization method andapparatus that make it possible to desorb and ionize sample ions underatmospheric pressure.

The present invention further provides a method and apparatus capable ofproducing the electrospray phenomenon even with regard to liquidbiological samples and samples having a high salt concentration.

The present invention further provides an ionization method andapparatus using electrospray in which dilution with a solvent is notalways necessary.

The present invention further provides an analyzing method and analyzingapparatus for analyzing ions that have been ionized by theabove-mentioned ionization method or apparatus.

An ionization method according to the present invention comprisesintroducing a sample into at least a tip portion of a hollow insulatedsample holder having a small hole in the tip portion; supporting anelectrically conductive linear body (inclusive of one that isneedle-shaped), which has been inserted inside the sample holder, suchthat a tip thereof is projectable outwardly from or retractable into thehole; projecting the tip of the linear body outwardly from the sampleholder through the hole while being brought into contact with the sampleinside the sample holder; and applying a high voltage to the linear bodyat least in a portion of a time period in which the tip of the linearbody is being projected outwardly from the hole, thereby ionizing, byelectrospray, the sample adhering to the tip of the linear body.

By using the sample holder, a sample can be taken directly at the tipportion thereof. A sample taken separately can also be introduced intothe tip portion of the sample holder as a matter of course. Anarrangement in which a liquid sample is supplied to the sample holderfrom liquid chromatography can also be adopted.

If necessary, a solvent may be supplied to a sample inside the sampleholder or to a sample adhering to the tip of the electrically conductivelinear body.

The application of a high voltage to the linear body at least in aportion of a time period in which the tip of the linear body is beingprojected outwardly from the hole includes the following modes ofimplementation: Specifically, a high voltage for electrospray may beapplied to the electrically conductive linear body constantly, or apulsed high voltage may be applied to the electrically conductive linearbody after the tip of the electrically conductive linear body isprojected outwardly from the hole of the sample holder. In the case ofthe latter, it is preferred that application of the pulsed high voltagebe halted when the sample at the tip of the electrically conductivelinear body has been consumed by electrospray. It is preferred that thehigh voltage be applied across the electrically conductive linear bodyand an ion introduction passage of an analyzing apparatus.

The small hole provided at the tip portion of the sample holder means asmall hole which, at its largest, will not allow a liquid sample, whichhas been introduced to the tip portion of the sample holder, to leak tothe exterior thanks to surface tension, or signifies a hole smaller thanthis.

In accordance with the present invention, the sample inside the sampleholder adheres to the tip of the electrically conductive linear bodywhen the tip of the electrically conductive linear body is projected(extended) outwardly from the hole at the tip of the sample holder. Whena high voltage is applied to the electrically conductive linear body,the sample adhering to the tip of the electrically conductive linearbody is ionized by electrospray. The ions are introduced to a massspectrometer and are analyzed.

In accordance with the present invention, ionization can be performedunder atmospheric pressure (in the atmosphere, in another inert gas orin a saturated vapor pressure chamber) without requiring that the sampleholder or electrically conductive linear body be placed in a vacuumchamber. The sample can be used as is without being subjected topre-treatment. It is possible to use a biological sample as the sample,and electrospray can be produced even with regard to samples having ahigh salt concentration. Furthermore, the sample need not necessarily bediluted using a solvent (the invention naturally does not excludedilution of the sample by a solvent). The diameter of the tip portion ofthe sample holder or of the electrically conductive linear body can bemade small, thereby enabling application to extremely small amounts ofsample, and it is also possible to improve the resolution of analysis.The electrically conductive linear body includes one obtained by coatingwith metal the surface of a needle made of an insulator such as glass(inclusive of quartz). If the diameter of the electrically conductivelinear body can be made smaller in this way, a much higher resolutioncan be obtained.

The present invention further provides an ionization analyzing method ofanalyzing molecules that have been ionized by the ionization methoddescribed above.

By repeating the projecting and retracting of the electricallyconductive linear body and the electrospraying of the sample multipletimes with regard to a single sample and trapping ions within theanalyzing apparatus or accumulating electric signals that are outputfrom the analyzing apparatus, the S/N ratio can be improved.

By using an electrically conductive linear body at least the tip ofwhich has been subjected to a hydrophobic or hydrophilic surfacetreatment, ion detection is possible in a time series in order ofincreasing surface activity with regard to all components havingdifferent surface activities within a liquid sample.

An ionization apparatus according to the present invention comprises: asupport mechanism for supporting a hollow insulated sample holder havinga small hole in a tip portion thereof; a driving unit for causing a tipportion of an electrically conductive linear body (inclusive of one thatis needle-shaped), which has been inserted inside the sample holder, toproject outwardly from or retract into the hole; and a high-voltagegenerating unit for applying a high voltage for electrospray to thelinear body at least in a portion of a time period in which the tip ofthe linear body is being projected outwardly from the hole.

The present invention further provides an ionization analyzing apparatushaving the above-mentioned ionization apparatus and an analyzingapparatus for analyzing ionized molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the overall configuration of an ionization apparatusand ionization analyzing apparatus (analytical apparatus) according toan embodiment of the present invention;

FIG. 2 is a sectional view illustrating an example of a sample holdersupporting unit;

FIG. 3 is a perspective view illustrating the manner in which cell fluidwithin a cell is collected as a sample;

FIG. 4, which illustrates the tip portion of the sample holder inenlarged form, shows a slender metal wire in the retracted state;

FIG. 5, which illustrates the tip portion of the sample holder inenlarged form, shows a slender metal wire in the projected (extended)state;

FIG. 6 illustrates another example of placement of a sample holder;

FIG. 7 illustrates another example of the configuration of a sampleholder;

FIG. 8 illustrates another example of the configuration of a sampleholder;

FIG. 9 is mass spectrum (graph) showing results of mass spectrometrybased upon ionization with regard to an insulin solution; and

FIG. 10 is mass spectrum (graph) showing results of mass spectrometrybased upon ionization with regard to a common onion.

DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 illustrates the general configuration of an ionization apparatusand ionization analyzing apparatus according to an embodiment of thepresent invention.

The ionization analyzing apparatus comprises an ionization apparatus 10and a mass spectrometry apparatus (ion analyzing apparatus) 30.

Sample ions desorbed and ionized from a sample by the ionizationapparatus 10 are introduced to the mass spectrometry apparatus 30.Although an (orthogonal) time-of-flight mass spectrometer can bementioned as an example of the mass spectrometry apparatus, the presentinvention is also applicable to a mass spectrometry apparatus such as a(linear) ion-trapping mass spectrometry apparatus, a quadrupole massspectrometry apparatus and a Fourier transform mass spectrometryapparatus. Further, an arrangement may be adopted in which anion-trapping apparatus is placed as a preceding stage to a spectrometryapparatus, such as a time-of-flight mass spectrometry apparatus, withthe exception of an ion-trapping mass spectrometry apparatus, ions thathave been ionized in the ionization apparatus 10 are accumulated by theion-trapping apparatus and the accumulated ions are subsequentlyintroduced to the mass spectrometry apparatus (the details will bedescribed later).

The interior of the mass spectrometry apparatus 30 is held in vacuum.The mass spectrometry apparatus 30 is equipped with a skimmer (orifice)31 for ion sampling, the tip of the skimmer has an ion introduction hole(ion introduction path) 31 a, and the interior of the mass spectrometryapparatus 30 is connected by the ion introduction hole 31 a to theoutside world (atmospheric pressure) in which the ionization apparatus10 has been placed. There is also an analyzing apparatus having an ionsampling capillary rather than a skimmer as the ion introduction path.Depending upon the type of mass spectrometry apparatus, there is a typein which an ion focusing voltage (a comparatively low voltage of lessthan +100 V in case of a positive-ion mode and less than −100 V in caseof a negative-ion mode) is applied to the ion sampling capillary(orifice) by a power supply unit. There are also cases where the ionsampling capillary is grounded. The outer wall of the mass spectrometryapparatus 30 generally is grounded.

The ionization apparatus 10 includes an XYZ stage 13; a supportmechanism 15 provided on the XYZ stage 13 and supporting a hollow sampleholder 11; a driving unit (actuator) 14 for driving a slender metal wire(an electrically conductive linear body) (inclusive of one having a tiptapered into the shape of a needle) 12 that has been inserted into thesample holder 11; and a high-voltage generating unit 17 for applying anelectrospray high-voltage (on the order of several kV to 1 kV or lessthan 1 kV) across the slender metal wire 12 and the skimmer 31 of themass spectrometry apparatus 30 (or ground or ground potential). Thedriving unit 14 is secured to the XYZ stage 13 by a support member 16.

In this embodiment, the electrically insulated sample holder 11 has theform of a glass capillary or pipette and has the shape of a slendercylinder overall, and comprises a central barrel portion having auniform diameter; a tapered portion (tapered distal end, tip,tip-constituting portion) formed to have a tapered shape so as to beincreasingly slender from the barrel portion to the tip 11 a; and a baseportion 11 b connected to the barrel portion on the side opposite thetapered portion. The tip (tip portion) 11 a of the sample holder 11 hasan opening (small hole) of very small inner diameter. As one example,the inner diameter of the hole in the tip 11 a may be 1 μm to severalhundred μm depending upon the type and size of the sample, although theinner diameter may be less than 1 μm.

As shown in FIG. 2, the sample holder 11 is supported in a horizontalattitude by the support mechanism 15 composed of two clamp plateserected vertically on the XYZ stage 13 with a small clearance betweenthem and formed to have arcuate recesses that clamp the barrel portionof the sample holder 11. The support mechanism 15 can be implemented invarious forms, such as by being constituted by a support column erectedon the XYZ stage 13 and an arm mounted on the upper portion of thesupport column and holding the sample holder 11, by way of example.

The slender metal wire 12 has been inserted into the sample holder 11from the base portion 11 b. It will suffice if the slender metal wire 12has a diameter sized such that the wire will pass loosely through thehole (opening) in the tip 11 a of sample holder 11, or if it has adiameter smaller than this. For example, the diameter of the slendermetal wire 12 is ½ to 1/100 of the inner diameter of the hole in the tip11 a, or smaller. The tip of the slender metal wire 12 may or may not betapered to a point. If necessary, a support member (e.g., a rubberstopper or the like having a center hole penetrated by the slender metalwire 12) (the support member preferably has an insulating property)supporting the slender metal wire 12 in a freely movable manner isplaced inside the sample holder 11 (e.g., inside the barrel portion).The tip portion of the slender metal wire 12 may be in contact with theinner surface of the tip 11 a (the inner side of the hole).

The driving unit 14 grasps the rear end portion of the slender metalwire 12 protruding outwardly from the base portion 11 b of sample holder11 and moves (displaces) (oscillates) (drives) the slender metal wire 12along the longitudinal direction thereof. As a result of this drivingoperation, the tip portion of the slender metal wire 12 is projected(extended) outwardly from or retracted into the hole in the tip 11 a ofsample holder 11.

The XYZ stage 13 supports the support mechanism 15 of the sample holder11 and the support member 16 of the driving unit 14 and displaces theseas a whole in three orthogonal directions, namely along the X, Y, Zdirections. For example, assume that the longitudinal direction of theslender metal wire 12 is the X direction. The position of the tip 11 aof sample holder 11 is adjusted by the XYZ stage 13 so as to be situatedin the proximity of, and external to along the X direction, the ionintroduction hole 31 a in the skimmer 31 of mass spectrometry apparatus30. Naturally, an adjustment may be made such that when the tip of theslender metal wire 12 has been projected (extended) from the tip 11 a ofthe sample holder 11, electrospray is generated without the tip of theslender metal wire 12 contacting the skimmer 31. The ionization of thesample is carried out under atmospheric pressure.

Preferably, the XYZ stage 13 and driving unit 14 include a device havinga movement function exhibiting good mechanical reproducibility, such asa piezoelectric element or a motor-driven or magnetically driven device,and can control the amount of displacement on the nm order along eachdirection. In particular, it is preferred that the driving unit 14,which reciprocates the slender metal wire 12 in the longitudinaldirection, be such that the frequency, amplitude and number ofoscillations of reciprocation (in which an oscillation includes a singlereciprocation) can be controlled.

A sample of interest is taken in the following manner, by way ofexample: As shown in FIG. 3, a portion L of an organism has been placedinside a laboratory dish 35. The sample holder 11 is attached to apiston syringe 19. The contents (cell fluid) of a specific single cell C(the diameter of which is on the order of 10 to 100 μm) within theportion L of the organism are drawn inside the tip 11 a (the interior ofthe tip or inside the tip portion) of sample holder 11 by the syringe19. Only the cell fluid (the target sample) of the single cell C istaken as a sample inside the tip portion of the sample holder 11.

If cell fluid (e.g., less than 100 fL) that is less than 10% of thetotal cell fluid of the single cell can be sampled under a microscope,this will circumvent the need to sacrifice the living cell. In thiscase, after the cell fluid is drawn in, the interior of the sampleholder (capillary) may be filled with a solvent (the solvent is drawnin) as necessary, thereby diluting the cell fluid sample (sampling ofthe sample multiple times need not be carried out). It is required thatthe tip of the sample holder be very slender, and it is required thatthe electrically conductive linear body inserted into the interiorthereof also be made very slender. As will be described later, insteadof a slender metal wire, an extremely slender electrically conductivelinear body can be obtained as the electrically conductive linear bodyif the surface of a slender glass wire (of diameter 1 to 100 μm, forexample) is coated with a metal (as by vapor-depositing the metal).

The sample holder 11 that has sampled the contents of the cell C isattached and secured to the support mechanism 15, as shown in FIG. 1.The slender metal wire 12 is inserted from the base portion 11 b of thesample holder 11. The tip of the slender metal wire 12 is situated onthe inner side of the tip portion of sample holder 11 (see FIG. 4).

The sample of interest is not limited to a cell. The sample method alsois not limited to that set forth above. In short, it will suffice if thesample to undergo mass spectrometry is introduced into the tip portionof the sample holder 11. The sample of interest may be a liquid orliquefied object. Owing to its surface tension, the liquid or liquefiedobject will not leak from the small hole in the tip 11 a of sampleholder 11. The tip (tip portion) of the slender metal wire 12 issituated inside the sample of interest (at the innermost position; topdead center or bottom dead center in accordance with the attitude of thesample holder).

In this state the slender metal wire 12 is projected (extended)outwardly from the hole of the end 11 a of the sample holder 11 (seeFIG. 5). As the tip (tip portion) of the slender metal wire 12 isprojected outwardly, the sample adheres to the tip (tip portion) of theslender metal wire 12 and is outwardly exposed. Owing to the surfacetension of the liquefied sample, the adhesion of the sample to the tip(tip portion) of the slender metal wire 12 is suppressed to the minimumextent, the coating thereof is substantially uniformalized and theamount of sample also is made substantially constant (meaning thatreproducibility is excellent).

The tip of the slender metal wire 12 moves through a predeterminedamount of displacement between the inward position (innermost positionor top dead center or bottom dead center) and outward position(outermost position or bottom dead center or top dead center) of the tipportion of sample holder 11. When the tip of the slender metal wire 12arrives at the outermost position (e.g., a position several tens ofmicrons to several millimeters distant from the tip 11 a), a pulsed highvoltage is applied across the slender metal wire 12 and skimmer 31. As aresult, the sample adhered to the tip (tip portion) of the slender metalwire 12 is ionized by electrospray. The ionized sample is introduced tothe interior of the mass spectrometry apparatus 30 from the introductionhole 31 a of the skimmer 31 and undergoes mass spectrometry.

Preferably, the in-and-out movement (the projecting and retracting) ofthe slender metal wire 12 from the tip 11 a of sample holder 11 and theapplication of the pulsed high voltage when the tip (tip portion) of theslender metal wire 12 is projected are repeated multiple times withregard to the same sample and ions are produced multiple times. In acase where the mass spectrometry apparatus is of the ion-trapping type,or if it is of the type having an ion-trapping apparatus provided as apreceding stage, the sample ions produced by the above-mentionedrepetition will be accumulated by ion trapping and, hence, a massspectrum having an excellent S/N ratio will be obtained. Further, a massspectrum having an excellent S/N ratio can be obtained by electricallyaccumulating (as by storing data in memory) an electric signal outputrepeatedly from the mass spectrometry apparatus owing to the repetitivegeneration of ions.

When the liquid sample is consumed by electrospray from the tip ofslender metal wire 12 and the metal surface of the slender wire becomesexposed, a gaseous discharge tends to occur. In such case, therefore,the voltage applied to the slender metal wire should be turned offbefore the discharge occurs. The pulse width of the voltage applied tothe slender metal wire in such case usually is less than 1 ms.

The high voltage for electrospray may be applied to the slender metalwire 12 continuously. In this case the electrospray would be producedwhen the tip of the slender metal wire 12 protrudes outwardly from thesample.

The voltage applied to the slender metal wire 12 is a positive highpotential in case of a positive-ion observation mode and is a negativehigh voltage in case of a negative-ion observation mode.

Although the sample holder 11 is arranged horizontally in FIG. 1, it mayalso be arranged vertically with the tip 11 a pointed downward, as shownin FIG. 6. Conversely, the sample holder 11 may be arranged verticallywith the tip 11 a pointed upward. The sample holder 11 may be arrangedat an incline. In any case, the sample holder 11 is placed at a positionwhere the sample ions resulting from electrospray produced from the tipof the slender metal wire 12 will be introduced efficiently into themass spectrometry apparatus 30 from the ion introduction hole 31 a. Itshould be noted that the high-voltage generating unit 17 is illustratedin simplified form in FIG. 6 and in the diagrams that follow it.

FIG. 7 illustrates a further embodiment. A sample holder 11A isconfigured in such a manner that a sample inflow passage 21 (a glasstube) is joined to the barrel portion of the above-mentioned sampleholder 11 so as to communicate with therewith. Further, a sample-outflowpreventing stopper 22 is used to plug the interior of the barrel portionon the base-end side of the portion to which the sample inflow passage21 is connected. The sample-outflow preventing stopper 22 is made of,for example, rubber. The slender metal wire 12 penetrates the stopper 22and extends up to the vicinity of the tip 11 a (outwardly or inwardlythereof) of sample holder 11A.

By way of example, the sample inflow passage 21 can be connected to theoutflow passage of liquid chromatography and the liquid outflow fromliquid chromatography can be introduced into the sample holder 11A sothat the liquid can undergo ionization and mass spectrometry.

FIG. 8 illustrates a further embodiment, in which a capillary 23 made ofglass is used as the sample holder and a tip portion 23 a thereof is cutoff at an angle. A plant or animal is pierced directly with theobliquely cut-off tip 23 a of the capillary 23 to thereby take a sample.If necessary, it can be arranged so that a solvent vapor is blown (thesolvent supplied) from a supply tube 24 toward the tip portion of thecapillary 23 when the sample is ionized. The mass spectrometry apparatusis not shown in FIG. 8.

It should be noted that an arrangement may be adopted in which thedriving unit 14 in FIG. 1 is omitted and the slender metal wire 12 ismoved in and out (extended and retracted) via an insulator manually bythe operator. Various modifications are conceivable. For example, theXYZ stage 13 is not necessarily required.

If the solvent is one that dissolves or moistens, any solvent may beused, and it may be in the form of a liquid or gas. Examples of thesolvent are water, alcohol, acetic acid, trifluoroacetic acid,acetonitrile, an aqueous solution, a mixed solvent and a mixed gas, etc.These solvents can be supplied to the tip of the sample holder in theform of a liquid as is or upon being converted to a mist or heated vaporor in the form of a gas.

Finally, ionization and result of analysis based thereon will beillustrated. FIG. 9 illustrates results obtained by using as the sampleholder a capillary (the diameter of which is constant over the entirelength) having an inner diameter of 250 μm, introducing an insulinsolution, which has a volume of about 0.03 μL (microliters), onto thetip portion of the capillary, inserting a tungsten wire, which has adiameter of 10 μm, as the slender metal wire, moving the tip portion ofthe wire in and out of the capillary tip and applying a high voltage of1.6 kV to thereby perform ionization and mass spectrometry byelectrospray.

FIG. 10 illustrates results obtained by piercing a common onion with acapillary (of inner diameter 250 μm) the tip of which has been cut offat an angle in the manner shown in FIG. 8, sampling the juice andinducing electrospray (at a voltage of 1.6 kV) by extending andretracting a tungsten wire having a diameter of 30 μm, therebyperforming ionization and mass spectrometry. Water vapor was used as thesolvent and this was sprayed toward the sample in the manner shown inFIG. 8. It will be understood that peaks appear for amino acids andsugars.

A body obtained by coating the surface (preferably the entire surface)of a linear body, which is made of an insulator such as glass (inclusiveof quartz) and capable of being finely extended, with a metal (as byvapor-depositing the metal to a thickness of approximately 0.1 μm orless) can be used as the electrically conductive linear body. Inaccordance with this mode of implementation, it is possible tomanufacture an electrically conductive linear body which is extremelyslender (having a diameter of less than 10 μm).

In a case where a liquid sample has been introducing into the interiorof the sample holder, an interface always exists between the solutionand the atmosphere. Components in the solution that exhibit highersurface activity are concentrated at this interface. Accordingly, ifthese components can be electrosprayed selectively, then all of thecomponents within the solution can be detected successively in order ofsurface activity. This becomes possible in accordance with the method ofthe present invention.

Specifically, the interior of a small sample holder (e.g., a capillaryhaving an inner diameter on the millimeter order) is filled with aliquid sample. A slender probe that has undergone a (hydrophobic)surface treatment for rendering the surface hydrophobic is insertedinside the sample holder. The hydrophobic surface treatment can beperformed as follows: For example, a titanium wire is exposed to aburner flame to thereby form an oxide film on the surface. This titaniumprobe is left standing in pentafluorophenyl-triethoxysilane (a 100% or50% methanol solution) for from several hours to twenty-four hours. Thesurface of the titanium probe is made hydrophobic as a result. Makingthe surface of a slender probe hydrophilic also is efficacious.

A high voltage is applied to the probe beforehand. Alternatively, a highvoltage is applied in pulsed fashion when the probe protrudes from thesurface of the liquid sample.

The probe that has been inserted into the sample holder is moved backand forth (extended and retracted) (e.g., at 3 Hz) along the axis(longitudinal direction) of the sample holder, the probe is made toprotrude forwardly from the liquid surface within the sample holder andthe liquid sample that has adhered to the probe tip is electrosprayedslowly.

By virtue of this operation, first ions of high interface (surface)activity that have condensed selectively at the liquid interface(surface) are electrosprayed. By repeating this operation, ions areelectrosprayed in order from ions of high to low surface activity. Thespectra vary with time from components of high surface activity tocomponents of low surface activity. Ions of all the analytes havingdifferent surface activities that are present in the liquid sample arethus detected.

With conventional electrospray, a liquid is fed through a capillary anda high voltage is applied to the capillary itself to therebyelectrospray the liquid. In this case, all components contained in theliquid are forcibly fed simultaneously and are electrosprayed. Hence,components exhibiting little surface activity are not released from thecharged droplets (they remain in the mother droplets during theoffspring droplets formation). With regard to such components,therefore, detection as gaseous-phase ions is difficult and detectionsensitivity is sacrificed. By contrast, in accordance with the presentinvention, a liquid sample is captured by a batch system within thesample holder and all droplets can be electrosprayed completely. Thatis, analysis of all components is possible. In particular, since aslender probe can be used (the tip diameter is less than onemicrometer), the amount of sample captured is small and detailedfractionation can be performed in order of surface activity to therebymake component analysis possible.

When a probe surface is made hydrophilic, hydrophobic sample ions areobserved. However, there are also cases where a hydrophilic sample willremain captured on the probe surface and will not be electrosprayed. Insuch cases ions will be observed if the probe tip is supplied with asolvent vapor to thereby promote electrospray. Accordingly, hydrophilictreatment is an excellent method of fractionating and electrosprayinghydrophobic ions and hydrophilic ions.

1-10. (canceled)
 11. An ionization method using electrospray comprising:introducing a sample into at least a tip portion of a hollow insulatedsample holder having a small hole in the tip portion; supporting anelectrically conductive linear body, which has been inserted inside saidsample holder, such that a tip thereof is projectable outwardly from orretractable into said hole; projecting the tip of said linear bodyoutwardly from the sample holder through said hole while being broughtinto contact with said sample inside said sample holder; and applying ahigh voltage to said linear body after the tip of said electricallyconductive linear body is projected outwardly from said hole of saidsample holder, thereby ionizing, by electrospray, the sample adhering tothe tip of said linear body.
 12. An ionization method according to claim11, wherein the projecting and retracting of the tip of saidelectrically conductive linear body and the electrospraying of thesample are repeated multiple times with regard to a single sample. 13.An ionization method according to claim 11, wherein application of saidhigh voltage is halted when the sample at the tip of the electricallyconductive linear body has been consumed by electrospray.
 14. Anionization method according to claim 11, wherein a sample is takendirectly at the tip of said sample holder.
 15. An ionization methodaccording to claim 11, wherein a liquid sample is supplied to saidsample holder from liquid chromatography.
 16. An ionization methodaccording to claim 11, wherein ionization is carried out underatmospheric pressure.
 17. An ionization method according to claim 11,wherein use is made to an electrically conductive linear body at leastthe tip of which has been subjected to a hydrophobic or hydrophilicsurface treatment.
 18. An ionization analyzing method of analyzingmolecules that have been ionized by the ionization method set forth inclaim
 11. 19. An ionization apparatus using electrospray, comprising: asupport mechanism for supporting a hollow insulated sample holder havinga small hole in a tip portion thereof; a driving unit for causing a tipportion of an electrically conductive linear body, which has beeninserted inside said sample holder, to project outwardly from or retractinto said hole; and a high-voltage generating unit for applying a highvoltage for electrospray to said linear body after the tip of saidelectrically conductive linear body is projected outwardly from saidhole of said sample holder.
 20. An ionization analyzing apparatus havingthe ionization apparatus set forth in claim 19 and an analyzingapparatus for analyzing ionized molecules.
 21. An ionization methodaccording to claim 12, wherein a sample is taken directly at the tip ofsaid sample holder.
 22. An ionization method according to claim 13,wherein a sample is taken directly at the tip of said sample holder. 23.An ionization method according to claim 12, wherein a liquid sample issupplied to said sample holder from liquid chromatography.
 24. Anionization method according to claim 13, wherein a liquid sample issupplied to said sample holder from liquid chromatography.
 25. Anionization method according to claim 12, wherein ionization is carriedout under atmospheric pressure.
 26. An ionization method according toclaim 13, wherein ionization is carried out under atmospheric pressure.27. An ionization method according to claim 12, wherein use is made toan electrically conductive linear body at least the tip of which hasbeen subjected to a hydrophobic or hydrophilic surface treatment.
 28. Anionization method according to claim 13, wherein use is made to anelectrically conductive linear body at least the tip of which has beensubjected to a hydrophobic or hydrophilic surface treatment.
 29. Anionization method according to claim 16, wherein use is made to anelectrically conductive linear body at least the tip of which has beensubjected to a hydrophobic or hydrophilic surface treatment.
 30. Anionization analyzing method of analyzing molecules that have beenionized by the ionization method set forth in claim 12.